The cytotoxicity and mutagenicity of the human carcinogen treosulphan, and its hydrolysis product, dl-1,2:3,4-diepoxybutane (DEB), were studied in AS52 cells, a PSV2gpt-transformed Chinese hamster ovary cell line. Factors such as the pH of the treatment medium, the duration of exposure, and the cell density during the treatments, were also investigated. Both chemicals were toxic and mutagenic. The toxicity and mutagenicity of treosulphan was pH-related, whereas the DEB effects were pH-independent. This finding supports the hypothesis that (pH-dependent) hydrolysis of treosulphan is necessary for the mutagenicity of this chemical. The molecular nature of the treosulphan and DEB-induced mutants are being investigated to ascertain whether both chemicals were mutagenic via the same ultimate mutagen. PCR analyses of restriction fragments from treosulphan and DEB-induced mutants showed similar patterns; 23% and 24% of the mutants carried point mutations, 26% and 21% had partial deletions of the targeted chromosome region, and 52% and 55% had complete deletions of this region, for treosulphan and DEB, respectively. These results suggest that the same molecular moiety is responsible for the mutagenicity of both chemicals. Studies are in progress to sequence the point mutations. Although metal salts are usually reported as nonmutagenic in cultured mammalian cells, preliminary studies have shown that a number of these salts are weakly mutagenic in the AS52 cells under the treatment conditions used in our laboratory. These mutagenic responses, and the effects of protocol variations on their presence and magnitude, are being further characterized. The mutants obtained will be sequenced to determine the precise modes of action of the different metals.